In many situations in clinical diagnostics, certain samples quantified by immunoassays can generate out of high range values, i.e. the analyte concentration is higher than the level where the assay can produce accurate and reproducible results. There is a need to quantify these samples because patients producing such out of high range analyte samples often have a high incidence of morbidity and mortality demanding medical attention. Immunoassays are also useful tools in these clinical situations not only for diagnosis but as well to monitor therapy.
Standard laboratory practice with an out of high range sample is for the laboratory personnel to further dilute the sample so that the analyte concentration falls within the quantification range followed by a repeat second assay. This protocol is problematic in that the re-assay requires more time to result, which can be detrimental in acute care or emergency rooms, and further incurs additional reagent cost to the laboratory. Some common lateral flow immunoassay devices do not have a means to dilute samples, which compels the user to perform the subsequent assay on another instrument enabling sample dilution protocols.
Solid phase immunoassays have limitations with high range samples because analyte concentrations can exceed the binding capacity of the immobilized antibody. Some clinical assays further require the combination of ultra sensitive detection of low analyte levels and quantification of high amounts of analyte, consequentially a wide quantification range is desirable. B-type natriuretic peptide, NTproBNP, and procalcitonin are such examples.
Development of solid phase immunoassays with a wide quantification range with low and high level detection are opposed technical goals. For example, in order not to exceed the immobilized antibody binding capacity with high analyte levels, samples are highly diluted ( 1/10- 1/100) or have short incubation times with the solid phase. Sensitive assays often require minimal sample dilutions (undiluted, ½-⅓) and relatively long incubation times with solid phase to effect binding of detectable amounts of trace analyte. The net result is often a compromise with less than adequate quantification range with either the low or high end of the analyte range having suboptimal clinical performance.
Arylsulfonate cyanine fluorescent dyes are described in Mujumdar et al. (1993) Bioconjugate Chemistry, 4:105-111; Southwick et al. (1990) Cytometry, 11:418-430; and U.S. Pat. No. 5,268,486. Cy5 is described in each of the references and is commercially available from Biological Detection Systems, Inc., Pittsburgh, Pa., under the tradename FLUOROLINK™ Cy5™. The arylsulfonate cyanine fluorescent dyes have high extinction coefficients (typically from 130,000 L/mole to 250,000 L/mole), good quantum yields, fluorescent emission spectra in a range (500 nm to 750 nm) outside of the autofluorescence wavelengths of most biological materials and plastics, good solubilities, and low non-specific binding characteristics.
Despite these excellent properties, arylsulfonate cyanine fluorescent dyes suffer from certain limitations. In particular, these dyes have a relatively narrow Stokes shift which results in significant overlap between the excitation and emission spectra of the dye. The overlap of excitation and emission spectra, in turn, can cause self-quenching of the fluorescence when the dye molecules are located close to each other when excited. Such self-quenching limits the number of arylsulfonate dye molecules which can be conjugated to a single antibody molecule for use in immunoassays. In the case of Cy5, an exemplary arylsulfonate cyanine fluorescent dye, the Stokes shift is 17 nm (which is the difference between an excitation wavelength of 650 nm and an emission wavelength of 667 nm). Optimal fluorescent yield is obtained when from two to four Cy5 molecules are conjugated to a single antibody molecule. The fluorescent signal output drops rapidly when more than four dye molecules are conjugated to a single antibody molecule. The inability to conjugate more than four dye molecules to individual antibody molecules significantly limits the sensitivity of immunoassays using Cy5-labelled antibodies and other binding substances.
U.S. Publication 2011/0312105 discloses a detection system and fluorescent immunoassays; the publication is incorporated herein by reference in its entirety.
There is a need for a method for quantitating an analyte having a wide range concentration in a single assay without having to dilute the sample and repeat the assay with fresh reagents.